The present invention concerns a method for the treatment of object surfaces prior to taking samples for microbiological investigations.
The invention concerns also a method for the pretreatment of object surfaces prior to cleaning.
The invention concerns also a test kit containing a sampler and a substance for the pretreatment of object surfaces.
Microorganisms, such as bacteria, yeast and mould fungi, attach readily to various surfaces. As they grow, they form layers called biofilms. There are various biofilms, often consisting of mixed populations of several different microbial species. The tendency of microbes to form biofilms can be seen as a survival strategy, through which they optimize the usage of the available nutrients. It is known that if microbes are allowed to form biofilms, it will be more difficult to cope with the health risks and pollution problems they cause. Within the shielding biofilm layer the microorganisms are able to efficiently resist the impact of antiseptics and antibiotics, and many patogenes are known to stay alive even for a long time with the protection of a biofilm (Costerton, J. W., Marrie. T. J. and Cheng, K.-J:, 1985. Phenomena of bacterial adhesion. In: Savage, D. C. and Fletcher, M. (Eds.) Bacterial Adhesion. New York: Plenum Press, p. 3-43).
Biofilms constitute a problem in many industrial fields. The problems they cause may be divided into two main groups: hygienic problems and problems affecting the production process per se. In the case of production that requires strict hygiene, even a slight microbial population may destroy the whole product. Slackened hygiene causes problems e.g. in food industry, in health care and in water supply systems. Other problems affecting the production process per se, such as formation of slime (mucus) in the apparatus and tubing, appear especially in wood and other processing industry, in air conditioning channels and even in desinfectant bottles. In some industrial areas microbes may even increase corrosion.
Biofilms formed by microorganisms, and the problems they cause, are, however, present also elsewhere than in industry, for instance on bathroom tiles, sauna benches, and in swimming and bubble pools.
If biofilms are allowed to form in food and process industry, the microbes they contain may pollute large amounts of product. Therefore hygienic monitoring is, or at least should be, used in most production plants. Often, however, the biofilm is found only when its become visible, and problems have already appeared, such as a functional disorder in the production unit, clogging of tubing or valves, or corrosion.
The biofilm consists mainly of water, 85-98%. In addition, the film contains polysaccharides and mineral collections formed by the microbes, and various dirt, depending on the environment (Costerton, J. W., Irvin, R. T. and Cheng, K.-J., 1981. The bacterial glycocalyx in nature and disease. Ann. Rev. Microbiol., 35, 299-324). When the circulation system works and water flows, the amount of biofilm is much larger than in the case of interrupted water circulation and an inspection of the dry tube surface. In a circulating liquid, a wet biofilm of 500 .mu.m, may shrink to 2-5% of its total volume and to a thickness of 10-25 .mu.m. Even if it is not necessarily visible, such a biofilm may increase friction and energy consumption in water circulation systems or in other tubings and plants. The structure of biofilms varies a lot, depending on the variation of surface materials, nutrients and microbes. This means also that there is no unique clear picture of their formation mechanism and exact structure. The biofilm structures must be determined separately for each case.
The biofilm can be found and studied by measuring its thickness and by dying the biofilm layer. Furthermore the microorganisms contained in the biofilm can be determined by taking a sample which is then cultivated on a growth medium.
The thickness of the biofilm is measured by various methods of analysis known as direct and indirect methods. The volumetric method is based on the determination of the wet weight, and the surface-mass-density methods are based on dry weight determinations. Furthermore the biofilms can be measured by microscopy, using light transmissivity, heat or electric conductivity, and oscillation. Most methods mentioned above are, however, difficult to perform in practical monitoring activities, because there are no user-friendly equipment (Characklis and Picologlou, 1978). Industry is interested in the source of the problem, not necessarily in the thickness of the biofilm.
Direct dying of biofilm on the surface of the tubing is potentially a good method to monitor its growth, but it is difficult to perform the dying in a closed tubing, because the dyes, often carcinogenic, enter the process, and the dyes per se may form precipitates, plus that they are difficult to remove. Therefore, it has been suggested that the biofilm be separated from its growing surface, after which the biofilm is dyed and its structure can be studied microscopically. In dying, it is possible to use various dyes, which attach to the polysaccaride chains, such as Alcian blue, toluide blue and erythrocine. From samples so dyed it has been found that biofilms that have been formed solely from microorganisms contain mainly polymers excreted by the microbes themselves, having structures consisting of complex polymers of various sugars.
Another method in use is the measurement of changes induced by the biofilm in heat conductivity and friction (Characklis, W. G. and Picologou, B. F. 1978. Measurement of the formation and destruction of primary biofouling films. In: Gray, R. H. (Ed.). Proceedings of the Ocean Thermal Energy Conversion (OTEC) Biofouling and Coosion Symposium. October 10-12, 1977. Seattle, Wash. Springfield Va., National Technical Information Service. p. 51-61.).
A monitor known as a biofilm monitor has been developed for finding and controlling biofilm. The monitor measures for example changes in pressure that may occur in a cooling equipment during flow. The change in pressure is proportional to friction, which, in its turn, is proportional to the friction of the cooling equipment. As in the indirect methods, such as those based on dying, inaccuracy has been a problem also of the monitors (Characklis, W. G. and Marshall, K. C. (Eds.). 1990. Biofilms. New York: John Wiley & Sons, Inc. 703 p.).
As mentioned above, the biofilms can be separated from the substrate by concentrated nitric acid In this case it is not possible to demonstrate and recognise the microorganisms by cultivation on a growthe medium, because said method destroys the microorganisms. The biofilm samples for microbial analysis have therefore in practice been taken with cotton swab, dipped in peptone saline solution, containing nutrients used by the microorganisms. Only small amounts of microorganisms can, however, be separated from the biofilm with the peptone saline solution, and the sampling surface still holds more than half of the biofilm after the swabbing.
The results above show that simpler and more user-friendly methods are needed for the detection of biofilms, especially in the process and air conditioning industry. In particular, methods are needed for checking whether the process surfaces are free from biofilms or not Biofilms constitute a special problem in the fields of industry mentioned above, and therefore the role of biofilms as a source of bacteria is being studied. In process hygiene, the assessment of a sufficient level of hygiene, and the attainment of this level, is of paramount importance. In the field of air conditioning there is a lack of knowledge concerning the influence of surface hygienic conditions on the amounts of microbes in the air.
Both in the process and air conditioning industry there is a need also to eliminate the disadvantages caused by the biofilms by cleaning and desinfecting the microbial growing surfaces, such as various tubing by strong desinfectants. Even strong desinfectants, however, are not necessarily able to remove the biofilms or to loosen their structures, so that the cleaning or desinfectant treatment would succeed.